The stated aspirational goal of the NEI, to Regenerate Neurons and Neural Connections in the Eye and Visual System, requires the development of modalities capable of non-invasively imaging neural connections as they are reestablished between the eye and the brain. In proof-of-concept studies we have introduced two promising techniques, diffusion basis spectrum imaging (DBSI) and diffusion functional magnetic resonance imaging (diffusion fMRI) for visualizing the pathology and function of the optic nerve in situ. In the current proposal, we will combine these technologies to deliver a new, diffusion MRI-based method to assess optic nerve anatomy, function and pathology simultaneously in both mice and human subjects. We will validate this approach by monitoring the progression and/or regression of axonal damage in glaucoma and optic neuritis. In keeping with the overall aspirations of the NEI, our long-term goal is to utilize this methodology to assess, non- invasively, the structure and function of regenerating axons in the optic nerve. Three specific aims will be pursued: (1) To quantify the relationships between diffusion MRI signals, axon number and visual function in an optic nerve crush mouse model, correlating DBSI with histological counts of axon number and diffusion fMRI with visual acuity; (2) To perform in vivo experiments and in silico computation (adapting structural information obtained from histology) on the optic nerve crush mouse model to identify a diffusion time optimized for both DBSI and diffusion fMRI and thus distinguish the contribution of restricted isotropic (distant from the axons) and anisotropic (adjacent to the axons) diffusion; and (3) To develop and optimize in vivo human optic nerve diffusion MRI protocol and visual stimulation paradigm that can simultaneously visualize optic nerve anatomy, function and pathology in glaucoma and optic neuritis patients. At completion, we will have established a novel imaging method to simultaneously assess optic nerve anatomy, function, and pathology allowing a detailed pathophysiological investigation of optic neuropathies.